The major goal of this proposal is to define the role of the nuclear transcription factor, C/EBPalpha in growth inhibition and immortalization. We have shown that C/EBPalpha inhibits cell proliferation in several transformed cell lines in vitro including human hepatoma cells and Saos2, a cell line that lacks p53 and Rb protein. We have further shown that induction of C/EBPalpha expression in stably transformed human fibrosarcoma cells inhibits cell growth and simultaneously elevates p21/SDI-1 mRNA and protein. Because the p21 protein is associated with cellular senescence in fibroblasts, we speculate that C/EBPalpha may be responsible, at least in part, for the resting or quiescent state of the normal hepatocyte in vivo. p21/SDI-1 mRNA is expressed in liver at levels that are easily detectable by Northern blotting. We will examine the role of C/EBPalpha in growth inhibition in three different ways. First, we will clone the target genes of C/EBPalpha that are involved in growth arrest in a novel cell system developed in our laboratory. We will use enzyme degrading subtractive hybridization to identify the genes that are targets of C/EBPalpha and that are responsible for growth arrest. Second, we will determine the biochemical basis for increased expression of p21/SDI-1 in cells that are growth arrested by C/EBPalpha. We will determine whether p21/SDI-1 expression is necessary and/or sufficient for growth arrest by blocking p21/SDI-1 expression using antisense strategies. Preliminary data suggest p53 and Rb are not required for growth arrest by C/EBPalpha; we will establish whether or not either of these tumor suppressor genes is involved in growth inhibition by C/EBPalpha. Finally, we will exploit a C/EBP knock- out mouse model that we have made in our laboratory. Cells derived from these animals will be cultured in vitro in order to test the hypothesis that the loss of C/EBPalpha will alter the growth properties or immortalization frequency of the cells.